Biomaterial Database

Alanine-scanning mutagenesis of the sixth transmembrane segment of gastric H+,K+-ATPase alpha-subunit. 2001

S Asano, and T Io, and T Kimura, and S Sakamoto, and N Takeguchi

Molecular Genetics Research Center and the Faculty of Pharmaceutical Sciences of Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan. shinji@ms.toyama-mpu.ac.jp

The sixth transmembrane (M6) segment of the catalytic subunit plays an important role in the ion recognition and transport in the type II P-type ATPase families. In this study, we singly mutated all amino acid residues in the M6 segment of gastric H(+),K(+)-ATPase alpha-subunit with alanine, expressed the mutants in HEK-293 cells, and studied the effects of the mutation on the functions of H(+),K(+)-ATPase; overall K(+)-stimulated ATPase, phosphorylation, and dephosphorylation. Four mutants, L819A, D826A, I827A, and L833A, completely lost the K(+)-ATPase activity. Mutant L819A was phosphorylated but hardly dephosphorylated in the presence of K(+), whereas mutants D826A, I827A, and L833A were not phosphorylated from ATP. We found that almost all of these amino acid residues, which are important for the function, are located on the same side of the alpha-helix of the M6 segment. In addition, we found that amino acids involved in the phosphorylation are located exclusively in the cytoplasmic half of the M6 segment and those involved in the K(+)-dependent dephosphorylation are in the luminal half. Several mutants such as I821A, L823A, T825A, and P829A partly retained the K(+)-ATPase activity accompanying the decrease in the rate of phosphorylation.

UI	MeSH Term	Description	Entries
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D002460	Cell Line	Established cell cultures that have the potential to propagate indefinitely.	Cell Lines,Line, Cell,Lines, Cell
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000409	Alanine	A non-essential amino acid that occurs in high levels in its free state in plasma. It is produced from pyruvate by transamination. It is involved in sugar and acid metabolism, increases IMMUNITY, and provides energy for muscle tissue, BRAIN, and the CENTRAL NERVOUS SYSTEM.	Abufène,Alanine, L-Isomer,L-Alanine,Alanine, L Isomer,L Alanine,L-Isomer Alanine
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D013270	Stomach	An organ of digestion situated in the left upper quadrant of the abdomen between the termination of the ESOPHAGUS and the beginning of the DUODENUM.	Stomachs
D013329	Structure-Activity Relationship	The relationship between the chemical structure of a compound and its biological or pharmacological activity. Compounds are often classed together because they have structural characteristics in common including shape, size, stereochemical arrangement, and distribution of functional groups.	Relationship, Structure-Activity,Relationships, Structure-Activity,Structure Activity Relationship,Structure-Activity Relationships
D013696	Temperature	The property of objects that determines the direction of heat flow when they are placed in direct thermal contact. The temperature is the energy of microscopic motions (vibrational and translational) of the particles of atoms.	Temperatures
D016296	Mutagenesis	Process of generating a genetic MUTATION. It may occur spontaneously or be induced by MUTAGENS.	Mutageneses
D017506	H(+)-K(+)-Exchanging ATPase	An enzyme isolated from the GASTRIC MUCOSA that catalyzes the hydrolysis of ATP coupled with the exchange of hydrogen and potassium ions across the cell wall. This enzyme was formerly listed as EC 3.6.1.36.	ATPase, Hydrogen, Potassium,Adenosinetriphosphatase, Hydrogen, Potassium,H(+)-K(+)-Transporting ATPase,Hydrogen, Potassium ATPase,Hydrogen, Potassium, Adenosinetriphosphatase,Adenosine Triphosphatase, Hydrogen, Potassium,Gastric H(+) K(+) ATPase,Hydrogen, Potassium, Adenosine Triphosphatase,Hydrogen-Potassium-Exchanging ATPase,Potassium Hydrogen ATPase,ATPase Hydrogen, Potassium,ATPase, Hydrogen-Potassium-Exchanging,ATPase, Potassium Hydrogen,Hydrogen Potassium Exchanging ATPase